How is potassium bromide used in infrared spectroscopy?
Sample preparation before conducting an infrared (IR) spectroscopy study is as critical as the study itself, and samples that are difficult to dissolve in any transparent IR solvent are mixed with sodium bromide potassium (KBr). This article examines the role of potassium bromide in infrared spectroscopy.
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The electromagnetic (EM) spectrum consists of seven different EM rays with wavelengths ranging from 10-12 at 103 M. Each type of radiation falls within a wavelength range that serves as a form of energy in scientific studies. The design of a spectrometer involves a specific EM radiation source absorbed by the sample and represented as a graph plotted for absorption versus wavelength.
IR spectroscopy measures the IR radiation absorbed by a chemical molecule of interest. Each chemical molecule has a set of functional groups that absorb IR radiation. Whatever the structure of the chemical compound, the functional groups tend to absorb IR radiation of the same frequency. The correlation between the molecular structure and the frequency at which it absorbs IR radiation allows the structural prediction of an unknown molecule.
In practice, low-energy mid-IR spectroscopy is preferable to high-energy near-IR (NIR) spectroscopy because mid-IR spectroscopy spectra come from simple vibrational modes of a molecule resulting in cleaner spectra.
Role of potassium bromide (KBr) in IR spectroscopy
Sample preparation before conducting an IR study is as critical as the study itself. Additionally, the medium used in sample preparation should be optically transparent in the IR region (4000-400 cm-1). In this context, alkali halides like potassium bromide (KBr) serve as a carrier with 100% transmittance. The major problem with mid-IR spectroscopy is the high absorbance of the radiation by the sample, and this can be solved by diluting the sample with spectroscopic grade KBr.
Samples that are difficult to dissolve in any IR transparent solvent are mixed with KBr in IR spectroscopy. The target sample to be analyzed is ground with KBr powder and pressed into a disk, also called a pellet. The size of the KBr particles plays an essential role in the preparation of the granules.
Sample preparation with KBr in IR spectroscopy
If the sample to be tested is of amorphous nature, it is dissolved in a non-aqueous volatile solvent and then deposited in the form of a thin layer of sample on the KBr cell by evaporation of the solvent.
For the KBr pellet method, it is essential to pulverize the KBr powder to a size of 200 mesh, followed by drying at 110°Celsius to remove any bound water molecules. For a sample pellet of 13 mm in diameter, 0.1 to 1% of the sample is mixed with 200 to 250 mg of powdered KBr. Next, the sample pellet is pulverized and placed in a pellet forming die. Additionally, an 8 ton force is applied under vacuum for several minutes to form a transparent sample pellet, and degassing removes air and bound water molecules.
In the diffuse reflectance method, KBr is packed in the diffuse reflectance accessory sample plate for background measurement, followed by dilution of the sample powder in KBr powder and packing of the resulting mixture in the sample plate for IR spectrum measurement.
In all of the above methods, it is essential to keep the sample and the KBr pellet away from moisture as the presence of water molecules gives a broad water peak at 3200 cm-1which overlaps the peaks of functional groups (amines, carboxylic acid, alcohol) in the range of 3500-3000 cm-1.
IR Spectroscopy Reveals Protein Structure in KBr Pellets – Research Studies
Fourier transform infrared spectroscopy (FTIR) is used to determine the secondary structure of protein in different physical states including solutions, suspensions, gels and solids. The most frequently used method in protein structure determination is to prepare a KBr pellet of freeze-dried protein.
In a study published in the Journal of Pharmaceutical Sciences, the authors determined the structure of two proteins, lysozyme and α-chymotrypsinogen, for the determination of the characteristic amide bond using FTIR. The results revealed that KBr treatment is a practical and viable option for determining dried protein structure and formulation.
In another study published in the journal Analytical biochemistry, the authors analyzed the secondary structure of 13 globular proteins in the KBr pellet by FTIR. The results showed a better correlation for the α-helix and the β-helix in the amide I band, where the secondary structure of the protein was retained in the solid state and packaged in a KBr pellet. Singular value decomposition (SVD) analysis showed that the absorbance of proteins in the KBr pellet was accurate compared to their absorbance in solution. In addition, the authors found that the secondary structure of proteins in the solid state is a better way to study water-insoluble grain proteins, fibers and storage proteins than their dissolution in organic solvents.
In another study published in Talanta, the authors developed a chemometric method to correct the spectra of the materials in the KBr disc by eliminating the interference from water. This is a common problem in the IR analysis of proteins and polysaccharides using the KBr disc technique. The resulting protein spectra were much more accurate than attenuated total reflection (ATR) spectra.
To summarize, KBr is optically transparent in the fingerprint region of IR spectroscopy and hence is used as a carrier in the form of a disk or pellet. Structural determination of sensitive biomolecules such as proteins has been possible using FTIR and the KBr disc technique, especially to determine their primary and secondary structures.
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References and further reading
Ingebrigtson, DN, and Smith, AL (1954). Infrared analysis of solids by the potassium bromide pellet technique. Analytical Chemistry, 26(11), 1765-1768. https://pubs.acs.org/doi/10.1021/ac60095a023
Forato, LA, Bernardes-Filho, R. and Colnago, LA (1998). Structure of proteins in KBr pellets by infrared spectroscopy. Analytical Biochemistry, 259(1), 136-141. https://pubmed.ncbi.nlm.nih.gov/9606154/
Ng, LM and Simmons, R. (1999). Infrared spectroscopy. Analytical Chemistry, 71(12), 343-350. https://pubs.acs.org/doi/abs/10.1021/a1999908r
Meyer, JD, Manning, MC and Carpenter, JF (2004). Effects of potassium bromide disc formation on infrared spectra of dried model proteins. Journal of Pharmaceutical Sciences, 93(2), 496-506. https://pubmed.ncbi.nlm.nih.gov/14705205/
Gordon, SH, Harry-O’kuru, RE and Mohamed, AA (2017). Elimination of Water Interferences in KBr Disc FT-IR Spectra of Solid Biomaterials by Resolved Chemometrics with Kinetic Modeling. Talanta, 174, 587-598 https://www.sciencedirect.com/science/article/pii/S0039914017306756